Estrous cycle influences the response of female rats in the elevated plus-maze test.

Abstract

The aim of this study was to examine the state of anxiety and the 17beta-estradiol and progesterone levels in rats tested in the elevated plus-maze during the four phases of the estrous cycle. Male rats, female rats during each of the four phases of the estrous cycle, ovariectomized rats, and diestrus female rats treated with estradiol were tested in the elevated plus-maze between 8:00 and 10:00 a.m. Blood was collected from all rats for the determination of 17beta-estradiol and progesterone levels. Female rats in the proestrus group spent more time in the open arms than diestrus rats (P<.05). There were no significant differences in the percentage of entries into the open arms or in the number of entries into the closed arms among the phases of the estrous cycle or between males and normal or ovariectomized females. Serum estradiol levels were higher (P<.05) during proestrus compared to estrus, metestrus, and diestrus in control and plus-maze tested female rats, but there were no significant differences in progesterone levels. Treating diestrus female rats with estradiol to produce estradiol plasma concentrations similar to those seen during proestrus abolished the difference in the percentage of time spent in the open arms by proestrus and diestrus rats. Since the time spent in the open arms of the plus-maze is inversely related to anxiety, we conclude that the anxiety levels of female rats were lower in proestrus than during diestrus, and that the levels of estradiol modulate this response.

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Estrous cycle influences the response of female rats in the elevated
plus-maze test
Fernanda Klein Marcondesa,*, Katia Jacqueline Miguelb, Liana Lins Melob,
Regina Ce ´lia Spadari-Bratfischb
aDepartamento de Cie ˆncias Fisiolo ´gicas, Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas (UNICAMP), Av. Limeira, 901, CEP
13414-018, Piracicaba, SP, Brazil
bDepartamento de Fisiologia e Biofı ´sica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, Sa ˜o Paulo, SP, Brazil
Received 29 June 1998; received in revised form 7 May 2001; accepted 22 June 2001
Abstract
The aim of this study was to examine the state of anxiety and the 17b-estradiol and progesterone levels in rats tested in the elevated plus-
maze during the four phases of the estrous cycle. Male rats, female rats during each of the four phases of the estrous cycle, ovariectomized
rats, and diestrus female rats treated with estradiol were tested in the elevated plus-maze between 8:00 and 10:00 a.m. Blood was collected
from all rats for the determination of 17b-estradiol and progesterone levels. Female rats in the proestrus group spent more time in the open
arms than diestrus rats (P<.05). There were no significant differences in the percentage of entries into the open arms or in the number of
entries into the closed arms among the phases of the estrous cycle or between males and normal or ovariectomized females. Serum estradiol
levels were higher (P<.05) during proestrus compared to estrus, metestrus, and diestrus in control and plus-maze tested female rats, but
there were no significant differences in progesterone levels. Treating diestrus female rats with estradiol to produce estradiol plasma
concentrations similar to those seen during proestrus abolished the difference in the percentage of time spent in the open arms by proestrus
and diestrus rats. Since the time spent in the open arms of the plus-maze is inversely related to anxiety, we conclude that the anxiety levels of
female rats were lower in proestrus than during diestrus, and that the levels of estradiol modulate this response. D 2001 Elsevier Science Inc.
All rights reserved.
Keywords: Estrous cycle; Anxiety; Estradiol; Progesterone; Elevated plus-maze test
1. Introduction
The influence of gender, reproductive cycle, and gonadal
hormones on many functions unrelated to reproduction has
been observed in humans and laboratory animals [2,35],
especially on the physiological responses to stressors
[20,40]. We have previously shown that footshock and
swimming stress alter the sensitivity to catecholamines of
right atria from female rats in diestrus, but not estrus
[20,38,39]. Basal anxiety levels can vary within an animal
depending on a variety of external and internal conditions
affecting both the neuropeptide and neurotransmitter levels
in the central nervous system. Therefore, the basal level of
anxiety may influence the perception of the challenge posed
by a stressor [14], modifying the stimulation extent of the
limbic system, sympathetic nerves, and hypothalamic–pitu-
itary–adrenal axis, depending on the gender and on the
estrous cycle phase.
Female rats spend more time in the open arms of the
plus-maze test than male rats, indicating a lower level of
anxiety in females [16]. Considering the effect of the
estrous cycle on the anxiety level, significant increases in
the time spent as well as in the percentage of entries into
the open arms during proestrus and estrus compared to
diestrus and metestrus have been found in literature [9].
However, the paper by Nomikos and Spyraki [30] shows
no significant difference between proestrus and diestrus
[30]. Under high light intensity, there is a slight increase in
the plus-maze open-arm exploration, during metestrus only
[24]. The administration of progesterone to ovariectomized
rats also increases open-arm exploration in the plus-maze
test under high light intensity [24]. Progesterone itself
0031-9384/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved.
PII: S0031-9384(01)00593-5
* Corresponding author. Tel.: +55-19-430-5380; fax: +55-19-430-5218.
E-mail address: fklein@fop.unicamp.br (F.K. Marcondes).
Physiology & Behavior 74 (2001) 435–440

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modulates anxiety in rats [33] and humans [35], and
fluctuations in estrogen and progesterone during the men-
strual cycle may be related to the depression and anxiety
susceptibility [34]. Such effects of steroid hormones on
neuronal activity can be explained by their capability of
modulating the neurotransmitter synthesis [17] and meta-
bolism [22], as well as their influence on the activity and
number of receptors for anxiolytic and anxiogenic agents
[4,5,19,34].
Based on the fact that the estrous cycle can influence
some rodent behaviors, that sex steroid levels vary during
the estrous cycle, and that steroid hormones can modulate
neuronal function, we have investigated the levels of anxiety
and the corresponding circulating concentrations of both
17b-estradiol and progesterone in female rats during the four
phases of the estrous cycle.
2. Method
2.1. Animals
Three-month-old male Wistar rats and female Wistar rats
in different stages of their estrous cycles were studied. An
additional group of female rats were bilaterally ovariectom-
ized under anesthesia (xylazine: 25 mg/kg and ketamine:
50 mg/kg im) 21 days before the experiment [28]. Another
group of female rats in diestrus was treated with estradiol
(50 mg/kg ip) 30 min before the elevated plus-maze test to
mimic the estradiol levels that occur during proestrus [27].
The rats were housed (five per cage) on a 12-h light/dark
cycle with lights on at 6:00 a.m. and were given free access
to food and water. All procedures were approved by the
university’s Animal Use Committee. The rats in all groups
underwent manipulation (males and ovariectomized
females) or estrous cycle determination (females) for at
least 10 days prior to testing.
2.2. Vaginal cytology
Estrous cycle phases were determined by vaginal lavage
[41] every morning between 7:00 and 8:00 a.m. and female
rats with at least two regular 4-day cycles were used.
2.3. Apparatus
The plus-maze [7,32] used in these experiments was
made of plywood and consisted of two open arms (50 cm
long?10 cm wide) and two closed arms (50 cm long?
10 cm wide, with 40 cm high walls) that extended from a
central platform elevated 50 cm above the floor.
2.4. Behavioral test
After determination of the estrous cycle phase (females)
or handling (males and ovariectomized females), 14–20 rats
representative of each of the seven experimental groups
were transferred to the experimental room. Thirty minutes
later, each rat was placed in the center of the plus-maze
[16,32]. The experimenter was in an adjacent room and
made the observations and recorded the behavior through a
one-way mirror located in the wall of the experimental
room. The number of entries into, and the time spent in
the open and closed arms was recorded over a 5-min period.
The maze was thoroughly cleaned after each test.
Each rat was tested only once. The three measures of
plus-maze behavior calculated were the percentage of ent-
ries into the open arms (100?open/total), the percentage of
time spent in the open arms (100?open/total), and the
number of entries into the closed arms. The percentage of
time spent in the open arms was interpreted as an index of
fear and anxiety, while the number of closed-arm entries
was an index of general activity [7,32]. The percentage of
open-arm entries is related to both anxiety and general
activity [7].
2.5. Blood sampling and hormone assays
Immediately after the elevated plus-maze test, the rats
were sacrificed in another room by a blow to the back of the
head followed by sectioning of the cervical blood vessels for
blood collection. Blood from control rats that were not
tested in the plus-maze (n=10–13/group) was obtained
30 min after the animals had been in the experimental room
for handling or estrous cycle determination, at the same time
of the day as the tested rats. Serum levels of 17b-estradiol
and progesterone were determined using commercial radio-
immunoassay kits (ICN Biomedicals Kit Nos. 07-270102
and 07-120102, respectively). Intra- and interassay coeffi-
cients of variation were <10%.
2.6. Statistical analysis
The results were expressed as the mean±S.E.M. Statist-
ical comparisons were done using analysis of variance
(ANOVA) followed by the Tukey’s test. Differences were
considered significant at P<.05.
3. Results
3.1. The elevated plus-maze
There were no differences in the performance of male
and female rats in the plus-maze test. Female rats in
proestrus spent a greater percentage of time in the open
arms than did females in diestrus, but there was no differ-
ence between proestrus and diestrus female rats treated with
estradiol [F(6,115)=3.00, P<.05; Fig. 1A]. There were no
significant differences in the percentage of open-arm entries
[F(6,115)=1.06, P>.05; Fig. 1B] or in the number of
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closed-arm entries [F(6,115)=1.27, P>.05; Fig. 1C] among
the groups studied.
3.2. Hormonal levels
The hormone levels in female rats during each of the four
phases of the estrous cycle are shown in Fig. 2. The serum
estradiol levels were 2.5–3.0-fold higher during proestrus
compared to estrus, metestrus, and diestrus in control
[ F(3,44) = 172.5, P < .0001] and plus-maze-tested
[F(3,70)=34.87, P<.0001] rats (Fig. 2A). In contrast, there
were no differences in the serum progesterone levels of
control [F(3,44) = 0.85, P>.05] and plus-maze-tested
female [F(3,70)=0.9623, P>.05] rats (Fig. 2B) in the four
phases of the reproductive cycle. There were also no
significant differences in the hormone levels between plus-
maze-tested rats and their respective controls (Fig. 2A and
B; Student’s t test, P>.05). The progesterone and 17b-
estradiol levels of male and ovariectomized rats were below
the detection limits of the radioimmunoassay used.
4. Discussion
The elevated plus-maze test is an experimental model
used to assess the state of anxiety in laboratory animals [32],
with the percentage of time spent in the open arms being
inversely related to anxiety [7,32]. Since the measurement
of anxiety may be influenced by locomotor activity, we
examined this factor by recording the absolute number of
closed-arm entries, considered a clear index of general
motor activity [7]. The higher percentage of time spent in
the open arms by proestrus and estradiol-treated diestrus
Fig. 1. Mean (±S.E.M.) percentage of the time spent in the open arms (A), percentage of the entries into the open arms (B), and the number of entries into the
closed arms (C) by male rats and different groups of female rats during the elevated plus-maze test. *P<.05, compared to diestrus. The number of rats per
group is indicated in parentheses.
Fig. 2. Serum estradiol (A) and progesterone (B) levels in control and plus-
maze tested female rats at different phases of the estrous cycle. The
columns represent the mean±S.E.M. The number of rats per group in the
plus-maze test was the same as in Fig. 1, and was 10–13 for the control
groups. *P<.05, compared to all the other groups (P<.05).
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female rats compared to diestrus female rats showed that the
level of anxiety was lower in proestrus. The lack of a
difference in the number of closed-arm entries among the
four phases of the estrous cycle indicated that the locomotor
activity of female rats in the plus-maze was not influenced
by the reproductive cycle. These findings indicated that the
anxiolytic effect during proestrus was indeed related to
anxiety and not to the locomotor activity of the rats.
Female rats have been reported to show a higher percent-
ageofentriesandtimespentintheopenarmsduringproestrus
and estrus than during diestrus under low-light condition
[24]. Higher time and percentage of entries into the open
arms during proestrus and estrus compared to metestrus and
diestrus have also been demonstrated [9]. On the other hand,
no differences have been observed between proestrus and
diestrus in another study [30]. In contrast, we have shown
here that there was a significant difference only between
the proestrus and diestrus rats. Differences in light intensity,
rat strain, and the period of data collection may explain the
divergent results of these studies. In this investigation, we
used Wistar rats and the experiments were performed 1.5–4
h after lights on, under regular illumination, whereas Diaz-
Veliz et al. [9] and Mora et al. [24] used Sprague–Dawley
rats and the observations were made 2–6 h after lights on
[9,24] or under dim illumination [24].
The influence of the estrous cycle on other behavioral
functions and the influence of hormones on the activity of
neuronal circuits involved in behavioral responses have
been reported [1]. The effects of sex steroids on behavioral
responses may be attributed to the organizational effects of
these hormones during the perinatal period and/or to their
cyclical variation in females during the estrous cycle. In the
present study, there were no differences between male and
female performances in the plus-maze test, indicating that
the lower level of anxiety during proestrus compared to
diestrus was probably related to the steroid hormone levels
during the estrous cycle. Estradiol levels were higher during
proestrus compared to the other phases. Moreover, the
behavior in the elevated plus-maze test of female rats in
diestrus that had been treated with an amount of estradiol
equivalent to that seen during proestrus [27] was similar to
normal female rats during proestrus. Thus, estradiol may be
related to the lower level of anxiety seen during proestrus.
The comparison between male and ovariectomized female
rats was not very informative since, although male and
ovariectomized female rats have low plasma levels of
estradiol, their overall hormonal profiles are completely
different from each other.
The activation of serotonergic receptors may result in
anxiogenic or anxiolytic responses that vary depending on
the receptor subtype, the brain structures activated, and the
drug dose. Most of the evidence suggests that an increase in
serotonergic activity has an anxiogenic effect in rats [13]. In
proestrus, the number of serotonin (5-HT) receptors is 50%
lower than in diestrus [3], and there is a decrease in the
concentrations of 5-HT in hypothalamic, limbic, and mid-
brain structures [17]. The uptake of 5-HT by the hypothal-
amus and the suprachiasmatic nucleus in vitro increases in
proestrus [23]. Together, these studies indicate that there is a
decrease in serotonergic activity during proestrus, and this
could explain the lower level of anxiety in this phase of the
estrous cycle. The mechanisms responsible for these
changes in serotonergic transmission are not yet fully
understood. The basal forebrain, considered to be the main
target of sex steroids in the brain, has a high expression of
estrogen receptors [4,25]. Steroid hormones exert their
actions through an interaction with gene expression and
regulation of protein synthesis [22,31]. Estradiol changes
the levels of progesterone [18] and oxytocin receptors [8]
and decreases 5-HT1receptor density [4] in the rodent brain.
Therefore, an estradiol-induced reduction in 5-HT synthesis,
in the number of 5-HT receptors, and/or an increase in 5-HT
uptake in the female rat brain could be related to the lower
anxiety seen in proestrus in the present study.
Substances other than 5-HT may also modulate anxiety.
The neurohypophyseal hormone oxytocin is associated with
a reduction in anxiety in mice, and this effect is enhanced by
estrogen [21]. Estradiol treatment selectively increases the
number of oxytocin binding sites in the ventromedial
nucleus and central amygdala of female rats [8], and this
phenomenon could be involved in the responses seen in the
present study.
Estradiol also modulates the excitability of neurons by
nongenomic mechanisms [26]. In the medial amygdala,
17b-estradiol directly changes the ionic conductance of the
postsynaptic membrane to produce a brief hyperpolarization
[29]. Classic anxiolytics, such as benzodiazepines, hyper-
polarize the postsynaptic membrane by acting on g-amino-
butyric acid (GABAA) receptors [15] to produce anxiolytic
responses. In addition, progesterone and estrogen have
additive effects that can increase the number of GABA
receptors in the brains of ovariectomized female rats by as
much as 160% [19].
Although we observed no differences in the progester-
one levels of rats during the estrous cycle, an influence of
progesterone in lowering the level of anxiety during
proestrus must be considered. The proestrus phase lasts
12–14 h and peripheral levels of progestins rise during the
afternoon of proestrus to reach peak levels at night [11].
Our results agree with a report [36] of low progesterone
levels between 8:00 and 10:00 a.m. on the morning of the
proestrus to estrus transition, with no significant differ-
ences among the estrous cycle phases. However, the rats
had been submitted to high levels of this hormone for
some hours during the night of proestrus, before the
experimental procedures. As a result, an influence of
progesterone on the levels of anxiety seen in our study
cannot be completely excluded. Progesterone metabolites
are important modulators of GABA receptors [5,6,12,
33,37], and an ovarian hormone-related change in the
sensitivity to the anxiolytic action of diazepam has been
reported in female rats [10].
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Although rats are nocturnal animals, they have been
studied extensively during the day light phase of the light/
dark cycle. Our results suggest that the influence of the
light/dark cycle and the hormonal fluctuations on the basal
differences in anxiety should be carefully considered in
studies of stress.
Our previous results showed that three footshock or
swimming stress sessions applied on consecutive days
during estrus, metestrus, and diestrus (diestrus group) pro-
duced subsensitivity to catecholamines in right atria that was
not altered when the stress was applied in diestrus, pro-
estrus, and estrus (estrus group) [20,38,39]. Based on
findings in the present study, it appears that this estrus
group received their second stress session when the anxiety
level of female rats is low. Different underlying levels of
anxiety during footshock or swimming sessions could affect
the activation of the adrenal glands, as well as the produc-
tion of gonadotrophins, sex, and thyroid hormones [14].
Since steroids can modulate adrenergic function [28], var-
iations in their levels during stress may differentially affect
the sensitivity to catecholamines (e.g., during diestrus, but
not estrus) in female rats submitted to footshock or swim-
ming sessions [20,38,39].
In conclusion, our results demonstrate that the state of
anxiety of female rats varies according to the estrous cycle,
with lower anxiety levels during proestrus than during
diestrus. Treating female rats in diestrus with estradiol
abolished this difference. Serum estradiol levels on the
morning of proestrus were higher when compared to those
for estrus, metestrus, and diestrus, but there was no change
in serum progesterone levels. Although we did not invest-
igate the mechanisms underlying the difference in anxiety
levels between proestrus and diestrus, 17b-estradiol is likely
to be involved in the anxiolytic response during the estrous
cycle in rats.
Acknowledgments
This research was supported by FAPESP (Grant No. 95/
3600-9). This is a part of a PhD thesis by Fernanda Klein
Marcondes done in the Departamento de Fisiologia e
Biofı ´sica, Instituto de Biologia, UNICAMP. The authors
thank Dr. Elenice Ferrari for the use of her laboratory and
Dr. Stephen Hyslop for editing the English.
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